The physiological and pharmacological effects of estradiol, the major ovarian estrogen, are well characterized. In particular, much research has been focused on the involvement of estradiol in endocrine-dependent carcinogenesis. However, less is known about the physiological and pharmacological activities of more than 20 metabolites of estrogen.
2-Methoxyestradiol is a naturally occurring mammalian metabolite formed by hepatic hydroxylation followed by O-demethylation of estradiol (Breuer et al. Naturwissenschaften 1960 12:280-81; Gelbke et al. J. Steroid Biochem. 1976 7:457-463). More research has been focused on this metabolite of estradiol as it has been found to be cytotoxic in cancer cell cultures. The cytotoxicity is associated with uneven chromosome distribution, faulty spindle formation, inhibition of DNA synthesis and mitosis, and an increase in the number of abnormal metaphases (Seegers et al. J. Steroid Biochem. 1989 32:797-809; Lottering et al. Cancer Res. 1992 52:5926-5923).
2-Methoxyestradiol also exerts a strong inhibitory effect on angiogenesis. Administration of 2-methoxyestradiol is thus suggested to be useful in the treatment of pathological angiogenesis and of angiogenic diseases such as angiogenic-sustained solid tumors. See U.S. Pat. No. 5,643,900.
In in vivo studies in mice, 2-methoxyestradiol was found to be a potent inhibitor of neovascularization and to inhibit their growth (Fotsis et al. Nature 1994 368:237-239). No signs of toxicity were observed in these animals.
2-Methoxyestradiol has also been reported to induce apoptosis in cancer cells. Specifically, it was found that 2-methoxyestradiol increases wild-type p53 levels in a human non-small cell lung cancer line associated with accumulation of the cyclin dependent kinase inhibitor p21WAF1/CIP1. See WO 98/042191. Significant apoptotic cell death was observed after drug treatment, thus indicating that 2-methoxyestradiol facilitates induction of p53-mediated apoptosis.
Various analogs of 2-methoxyestradiol have been prepared.
For example, Cushman et al. synthesized an array of 2-methoxyestradiol analogs to define the structural parameters associated with the antitubulin activity and cytotoxicity of this estradiol metabolite (J. Med. Chem. 1995 38(12) :2041-2048). 2-Ethoxyestradiol and 2-((E)-l-propenyl)-estradiol were disclosed as being substantially more potent than 2-methoxyestradiol itself. Analogues which inhibited tubulin polymerization also displayed significantly higher cytotoxicity in the MDA-MB-435 breast cancer cell line than in the other cell lines studied. Potencies of these analogues as cytotoxic and mitotoxic agents in cancer cell cultures correlated well with their potencies as inhibitors of tubulin polymerization.
D""Amato et al. (U.S. Pat. No.5,892,069) discloses a number of estrogenic compounds similar in structure to 2-methoxyestradiol for use as antimitotic agents. Various substitutions of the methoxy at position 2 are disclosed.
In the present invention compositions are provided which comprise a modified 2-alkoxyestradiol structure wherein all of the hydrogens of the carbon next to the oxygen of the alkoxy moiety at position 2 are substituted with a halogen or alkyl group. These modifications in the compositions of the present invention are believed to prevent or inhibit their O-dealkylation thereby extending their pharmacological activity.
An object of the present invention is to provide a composition comprising a compound of formula (I): 
wherein R1, R2 and R3 are each selected from the group consisting of halogens, methyl or longer chain alkyl groups or aromatic groups, and trifluoromethyl groups.
Another object of the present invention is to provide a method of prolonging inhibition of microtubule formation and tubulin polymerization and/or depolymerization via a composition of formula (I).
Another object of the present invention is to provide a method for treating diseases characterized by abnormal cell mitosis via administration of a composition of formula (I). Compositions of the present invention are believed to be particularly useful in treating neoplastic diseases including cancer.